Alcohol-related health problems are the worldwide health concern. More than 50% of Veterans use alcohol and nearly 23% are binge or heavy drinkers. Pathogenesis of alcoholic liver disease (ALD) involves endotoxemia caused by gut barrier dysfunction. About 25% of alcoholics develop ALD, but the factors that make them susceptible to ALD remain to be determined. The relationship between stress and alcoholism is well known, so this project focuses on the effects of stress on alcoholic tissue injury. Our preliminary studies showed that different types of stress disrupt tight junctions (TJs) and increase permeability in the intestinal epithelium by a mechanism that involves intracellular signaling elements. Preliminary data also indicated that chronic restraint stress exacerbates alcohol-induced gut permeability and associated liver injury. Probiotics are well known to promote gastrointestinal cell survival and barrier function. Our preliminary data indicated that Lactobacillus casei (L. casei) ameliorates stress and ethanol-induced gut barrier dysfunction. On the basis of these results it is hypothesized that: a) stress-induced cell signaling promotes alcohol-induced TJ disruption and barrier dysfunction in the intestinal epithelium, b) chronic stress exacerbates ethanol-induced gut barrier dysfunction, endotoxemia and liver injury by a CaV1.3 channel and JNK2-dependent mechanism, and c) L. casei ameliorates stress and ethanol-induced gut barrier dysfunction, endotoxemia and liver injury. Using a cell culture model of the intestinal epithelium and gene knock out mice we will determine that: 1) Ca2+-induced mitochondrial oxidative stress mediates stress-induced activation of Ask1/MKK7/JNK2/c-Src signaling and TJ disruption in the intestinal epithelium, 2) Glucocorticoids mediate stress-induced cell signaling in the intestinal epithelium and exacerbation of alcoholic gut permeability, 3) Stress-activated signaling promotes acetaldehyde-induced TJ disruption, 4) CaV1.3 channel and [Ca2+]i mediate the synergistic effects of chronic stress and ethanol on gut and liver, 5) Down regulation of JNK2 blocks stress and alcohol-induced gut barrier dysfunction and liver injury, 6) L. casei prevents stress-induced cell signalin and attenuates acetaldehyde-induced TJ disruption in Caco-2 cells and mouse intestine ex vivo, 7) L. casei attenuates stress and ethanol- induced gut permeability and liver injury in vivo, and 8) Peptide-like factor released by L. casei blocks gut barrier dysfunction and prevents liver injur caused by stress and ethanol. The outcome of these studies will have direct impact on the fields of stress-induced alteration of intestinal mucosal homeostasis as well as pathogenesis of alcoholic liver disease. .